MAMMALIAN THIOREDOXIN REDUCTASE IS IRREVERSIBLY INHIBITED BY DINITROHALOBENZENES BY ALKYLATION OF BOTH THE REDOX-ACTIVE SELENOCYSTEINE AND ITS NEIGHBORING CYSTEINE RESIDUE

The immunostimulatory dinitrohalobenzene compound 1-chloro-2,4-dinitro benzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase ( TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arner, E. S. J., Bjornstedt, M., and Hohmgren, A. (1995) J. Biol. Chem. 270, 3479-3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immu nomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenopr otein. We found that 4-vinylpyridine competed with DNCB for inactivati on of TrxR, with DNCB being about 10 times more efficient, and only al kylation with DNCB but not with 4-vinylpyridine induced an NADPH oxida se activity. A number of nonsensitizing DNCB analogues neither inactiv ated the enzyme nor induced any NADPH oxidase activity. The NADPH oxid ase activity of TrxR induced by dinitrohalobenzenes generated superoxi de, as detected by reaction with epinephrine (the adrenochrome method) . Addition of superoxide dismutase quenched this reaction and also sti mulated the NADPH oxidase activity. By peptide analysis using mass spe ctrometry and Edman degradation, both the cysteine and the selenocyste ine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alk ylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, inv olving a functional FAD in the alkylated TrxR generating an anion nitr oradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibi ted reduction of thioredoxin by the modified thioredoxin reductase aft er reaction with dinitrohalobenzenes may play a major role in the infl ammatory reactions provoked by these compounds.